Full bacterial genome assembled from scratch

In a big step towards an engineered genome, researchers managed to assemble an …

Rumors have been circulating for a while that Craig Venter, who developed shotgun sequencing, was poised to announce completion of his next goal: the creation and use of an entire genome from scratch—scratch being chemical bottles containing the four bases that are the basic building blocks of DNA. We're not quite there yet, but an advanced publication released by Science today takes us nearly to the finish line. It describes the assembly of a bacterial genome, all 582,970 bases of Mycoplasma genitalium, from basic chemicals. Right now, however, that genome resides in a yeast and hasn't been moved back into a bacteria.

The process of synthesizing kilobase-sized stretches of DNA from chemicals has become so efficient that the authors didn't even need to do this step themselves; they ordered out for it in the same way that you might order custom printing on a shirt. In fact, they later caught an error in the sequence that they traced back to a mistake on the order form.

People have assembled this sort of synthetic DNA into smaller, viral genomes before, but no one had tried to piece together something hundreds of kilobases long. The paper describes a hierarchal method that did much of the assembly in parallel and in test tubes. The researchers ordered up the sequence in chunks of six kilobases, with neighboring chunks having small regions of overlap. These overlaps then allowed them to combine sets of four chunks, along with a backbone for a bacterial artificial chromosome (BAC) in a single reaction. The resulting BAC and its 24-kilobase insert grew happily in E. coli cells.

A simple digest popped these fragments out of the BAC, and a similar process allowed two more assemblies, bringing the size of the BACs up to over 200 kilobases. That's where the technique ran into problems, as BACs don't work well with that much DNA. To avoid this problem, the researchers turned to YACs, or yeast artificial chromosomes. In a clever trick, the researchers let yeast do the hard work for them. Putting linear BAC DNA into yeast triggered the yeast's DNA repair machinery, which recognized and recombined the BAC DNA at overlapping sequences. Nearly 20 percent of the constructs recovered in yeast contained the full-length synthetic genome.

Why go through all this work just to recreate a genome that already exists? The key feature of this method appears to be that every single intermediate piece of the genome now exists in the freezers at the J. Craig Venter Institute. At their leisure, the authors can redo the assembly with some genes missing, or with new genes inserted from other organisms. Ultimately, they hope to get a better sense of both the basic metabolism of a simple organism, and begin to engineer that metabolism to do useful things. The authors will now have to perform these sorts of experiments in order to demonstrate that this method really is better than simply using the normal genome as a starting point.

The paper contains an interesting discussion that suggests that this method might be somewhat limited. Mycoplasma genitalium uses a slightly different genetic code from E. coli and yeast. Its code inserts a histidine where the code in those other organisms calls for a stop to protein production. This means that the BAC and YAC constructs aren't likely to make any Mycoplasma proteins in those organisms, preventing the constructs from interfering with normal metabolism. Trying to do the same things using organisms with an identical genetic code is unlikely to work as well.